BU-3839T antibiotic

ABSTRACT

A new antibiotic having the chromophore structure of the plurmycin family of antibiotics is produced by fermentation of Streptomyces violaceus ATCC 53807. The new antibiotic designated BU-3839T, exhibits potent antibacterial activity and also inhibits the growth of tumors in experimental animal systems.

This is a Division of our prior co-pending application Ser. No. 246,393filed 09/19/88.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a novel antibiotic compound designatedBU-3839T. BU-3839T exhibits antibacterial activity againstgram-positive, gram-negative and anaerobic bacteria and alsodemonstrates both in vitro and in vivo antitumor activity.

2. Description of the Prior Art

Structural studies done on BU-3839T indicate that it contains thechromophore, 11-hydroxy-5-methyl-4H-anthra[1,2-b]pyran-4,7,12-trione,found in the pluramycin family of antibiotics. Four antibiotics withthis chromophore have been reported, α-indomycinone (J. Antibiotics, SerA, 9, 75-81, 1956), SF-2330 (J. Antibiotics 39: 780-783, 1986),SS-43405D (Japan Kokai 61-139,394, June 26, 1986) and SS-43405E (JapanKokai 61-189,280, Aug. 22, 1986). α-Indomycinone was reported to have noantimicrobial activity, while SF-2330 and SS-43405D were shown to haveantibacterial activity. No antitumor activity has been reported for anyof the above-mentioned antibiotics.

The structures of α-indomycinone, SF-2330, SS-43405D, SS-43405E, andBU-3839T are shown below. BU-3839T can be distinguished from the otherantibiotics by the unique alkyl side chain (R₁) at the C-2 position ofthe chromophore.

    ______________________________________     ##STR1##    R.sub.1                 R.sub.2    ______________________________________    α-Indo- mycinone             ##STR2##           CH.sub.3    SF-2330             ##STR3##           CH.sub.3    SS-43405D             ##STR4##           CH.sub.2 COOH    SS-43405E             ##STR5##           CH.sub.3    BU-3839T             ##STR6##           CH.sub.3    ______________________________________

SUMMARY OF THE INVENTION

The present invention provides the antibiotic BU-3839T which exhibitsactivity against a variety of gram-positive, gram-negative and anaerobicbacteria. Additionally, BU-3839T exhibits in vitro and in vivo antitumoractivity.

BU-3839T is obtained by cultivating a BU-3839T-producing strain ofStreptomyces violaceus in an aqueous nutrient medium containingassimilable sources of carbon and nitrogen under submerged aerobicconditions until a substantial amount of BU-3839T is produced by saidorganism in said culture medium and then recovering the BU-3839T fromsaid culture medium.

In another aspect there are provided pharmaceutical compositions usefulfor treating bacterial or carcinostatic infections in a mammalian hostcomprising an effective bacterial-inhibiting or tumor-inhibiting amountof BU-3839T together with a pharmaceutically acceptable carrier.

In a further aspect the present invention provides a method of treatingbacterial infections in an animal host by administering to said host aneffective antibacterial amount of BU-3839T, or a pharmaceuticalcomposition thereof.

Finally the present invention provides a method of inhibiting the growthof tumors in a mammalian host by administering to said host atumor-inhibiting amount of BU-3839T, or a pharmaceutical compositionthereof.

DETAILED DESCRIPTION

The BU-3839T antibiotic of the present invention is produced byfermentation of a BU-3839T-producing strain of Streptomyces violaceus.The preferred producing organism is a novel strain of Streptomycesviolaceus designated herein as Streptomyces violaceus strain P950-4.This strain was isolated from a soil sample collected in Hyderabad,Andhra Pradesh State, India. A biologically pure culture of strainP950-4 has been deposited with the American Type Culture Collection(ATCC), Washington, D.C. and added to its permanent collection ofmicroorganisms as ATCC 53807.

Strain P950-4 has the following properties:

Morphology

Strain P950-4 forms both substrate and aerial mycelia, which are long,well-branched and not fragmented into short elements. Chains of sporesare formed on the monopodially branched aerial hyphae. The spore chainsare loop or compact spiral in shape, and contain 10 to 50 spores perchain. The spores are spherical to oval (0.6×0.6-0.9 μm) and have spinysurface. Motile spores and sporangium-like bodies are not observed.

Cultural Characteristics (Table 1)

The aerial mycelium are well formed on most agar media, but poorly onglucose-asparagine agar and not on ISP medium No. 6. The color of aerialmycelium is white, later turning to light grayish pink with sporulation.Melanin is produced distinctly in ISP medium No. 6, poorly in ISP No. 1,but not in ISP No. 7. A reddish-orange to purplish-red pigment, which ismore or less diffusible, is formed in ISP media Nos. 2 and 7, Bennett'sagar and Czapek's sucrose-nitrate agar. The pigment is pH-sensitive(yellowish-orange in acid and violet in alkali).

Physiological Characteristics (Table 2)

The growth is observed between 17° C. and 40° C., but not at 15° C. and43° C. Tyrosinase reaction is positive. All of eleven diagnostic sugarsare utilized for growth.

                                      TABLE 1    __________________________________________________________________________    Cultural characteristics of Strain P950-4                   Growth    Medium         Aerial mycelium Substrate mycelium                                                  Diffusible    __________________________________________________________________________                                                  pigment    Sucrose-nitrate agar                   Good            Light yellowish brown (76)                                                  Brownish orange (54)    (Czapek-Dox agar)                   Moderate, white (263)                                   to vivid dark purplish red                                   (260)    Tryptone-yeast extract broth                   Moderate, not turbid                                   Colorless      Deep brown (56)    (ISP No. 1)    Yeast extract-malt extract                   Good            Pale yellow (89)                                                  Strong yellowish brown    agar (ISP No. 2)                   Good; grayish yellowish pink                                   to brownish orange (54)                   (32)    Oat meal agar (ISP No. 3)                   Moderate        Pale yellow (89)                                                  None                   Moderate; grayish pink (8)    Inorganic salts-starch agar                   Moderate        Deep yellow (85)                                                  None    (ISP No. 4)    Moderate; grayish pink (8)    Glycerol-asparagine agar                   Moderate        Strong yellowish brown (74)                                                  Moderate yellow (87)    (ISP No. 5)    Moderate; grayish pink (8)    Peptone-yeast extract-iron                   Moderate        Colorless      Brownish black (65)    agar (ISP No. 6)                   None    Tyrosine agar (ISP No. 7)                   Moderate        Light olive brown (94)                                                  Dark grayish yellow (91)                   Moderate; yellowish white (92)    Glucose-asparagine agar                   Poor            Pale yellow (89)                                                  None                   Scant; white (263)    Nutrient agar  Poor            Colorless      Dark brown (59)                   None    Bennett's agar Good            Deep yellowish brown (75)                                                  Moderate yellowish brown                                                  (77)                   Moderate; white (263)    Papavizas' V-8 juice-                   Moderate        Dark grayish reddish brown                                                  Deep brown (56)    dextrose-yeast extract agar                   Moderate; grayish yellowish                                   (47)                   pink (32)    __________________________________________________________________________     Observation after incubation at 28° C. for 3 weeks.     Color and number in parenthesis follow ISCCNBS designation.

                  TABLE 2    ______________________________________    Physiological characteristics of Strain P950-4                          Utilization                          of**    ______________________________________    Hydrolysis of:    Gelatin        +            Glycerol   +    Starch         +            D-Arabinose                                           -                                L-Arabinose                                           +    Milk coagulation                   +            D-Xylose   +    peptonization  +            D-Ribose   +                                L-Rhamnose +    Production of:              D-Glucose  +                                D-Galactose                                           +    Nitrate reductase*                   +/+          D-Fructose +    Tyrosinase     +            D-Mannose  +                                L-Sorbose  -    Tolerance to:               Sucrose    +                                Lactose    +    Lysozyme   0.01%   ±         Cellobiose                                             +               0.001%  ±         Melibiose                                             +               None    +            Trehalose                                             +    NaCl       1-7%    +            Raffinose                                             +               8%      -            D-Melezitose                                             +    pH                 4.3 ˜ 10.5                                    Soluble  +                                    starch                                    Cellulose                                             -    Temperature:                Dulcitol   -                                Inositol   +    Growth range   17-40° C.                                D-Mannitol +    Optimal growth 33-38° C.                                D-Sorbitol ±    No growth      12° C. and 43° C.                                Salicin    ±    ______________________________________     *Czapek's sucrosenitrate broth/peptonenitrate broth     **Basal medium: PridhamGottlieb's inorganic medium (ISP Medium No. 9)

Cell Chemistry

The whole cell hydrolysate contains LL-diaminopimelic acid, glucose andribose, and hence the cell wall belongs to Type I and the sugar patternNC. The phospholipid contains diphosphatidylglycerol,phosphatidylethanolamine and phosphotidylinositol, and therefore isplaced in Type P-II.

Taxonomic Position

The morphology, cultural and physiological characteristics and cellchemistry reveal that Strain P950-4 is a species of Streptomyces.According to the descriptions of Pridham and Tresner.sup.(1), the majorcharacteristics of Strain P950-4 are as follows: (A) color of sporulatedaerial mycelium, red (R), (B) spore-chain morphology, Spira (S), (C)melanoid pigments, positive (C⁺), (D) spore wall ornamentation,echinulate (spiny). A species group with the above properties is shownin Table 17.44e (page 812), in which nine species and two subspecies aredescribed..sup.(1) These species and subspecies are fairly related toeach other. According to the descriptions of Shirling andGottlieb.sup.(2), Strain P950-4 is closely related to

It is to be understood that the present invention is not limited to useof the particular preferred Strain P950-4 described above or toorganisms fully answering the above descriptions. It is especiallyintended to include other BU-3839T-producing variant or mutant strainsof the said organism which can be produced by conventional means such asx-radiation, ultraviolet radiation, treatment with nitrogen mustards,phage exposure, and the like.

Preparation of BU-3839T

BU-3839T is produced by cultivating a BU-3839T-producing strain ofStreptomyces violaceus, most preferably a strain having thecharacteristics of Streptomyces violaceus strain P950-4 (ATCC 53807) ora variant or mutant thereof, under submerged aerobic conditions in anaqueous nutrient medium. The producing organism is grown in a nutrientmedium containing an assimilable carbon source, for example L-arabinose,D-xylose, D-ribose, D-glucose, D-fructose, sucrose, lactose, cellobiose,D-mannitol or soluble starch. The nutrient medium should also contain anassimilable nitrogen source such as fish meal, peptone, soybean flour,peanut meal, cottonseed meal or corn steep liquor. Nutrient inorganicsalts can also be incorporated in the medium. Such salts may compriseany of the usual salts capable of providing sodium, potassium, ammonium,calcium, phosphate, sulfate, chloride, bromide, nitrate, carbonate, orlike ions.

Production of BU-3839T can be effected at any temperature conducive tosatisfactory growth of the organism, e.g. 17°-40°, and is convenientlycarried out at a temperature of about 28° C.

The fermentation may be carried out in flasks or in laboratory orindustrial fermentors of various capacities. When tank fermentation isto be used, it is desirable to produce a vegetative inoculum in anutrient broth by inoculating a small volume of the culture medium witha slant or soil culture or a lyophilized culture of the organism. Afterobtaining an active inoculum in this manner, it is transferredaseptically to the fermentation tank medium for large scale productionof BU-3839T. The medium in which the vegetative inoculum is produced canbe the same as, or different from, that utilized in the tank as long asit is such that a good growth of the producing organism is obtained.

In general, optimum production of BU-3839T is achieved after incubationperiods of about 2-3 days. Antibiotic production can be monitored by thepaper disc-agar diffusion assay using Bacillus subtilis M45(rec⁻) as thetest organism and in vitro cytotoxic activity against B16 melanomacells.

Isolation and Purification

BU-3839T may be isolated from the fermentation broth by conventionalisolation and purification procedures, i.e. solvent extraction andchromatography. Example 2 below illustrates a typical isolation andpurification procedure for obtaining BU-3839T in substantially pureform.

Physicochemical Properties

BU-3839T was obtained as yellowish-orange needles which melted at 255°C. with decomposition. It was soluble in dimethyl sulfoxide andN,N-dimethylformamide, slightly soluble in chloroform, methylenechloride and alkaline water, and almost insoluble in other organicsolvents and water. It gave a positive response to ferric chloridereagent, but gave no coloration with ninhydrin, anthrone and Sakaguchitests. The physicochemical properties of BU-3839T are summarized belowin Table 3. The molecular formula of the antibiotic was established asC₂₂ H₁₆ O₈ by the mass spectrum (M⁺ m/z:408) and microanalysis. BU-3839Texhibited UV absorption maxima at 240, 267, 286 (shoulder) and 417 nm inmethanol and acidic methanol which shifted to 243, 282 (shoulder), 324and 518 nm in alkaline solution. The IR spectrum showed characteristicabsorptions at 1670, 1650, 1640, 1630 and 1580 cm⁻¹ suggesting a quinonemoiety.

                  TABLE 3    ______________________________________    Physicochemical Properties    ______________________________________    Nature          Yellowish-orange needles    M.P.            255° C.(dec.)    [α].sub.D 25.0                    +26° ± 4 (c 0.25, DMF)    UV λ.sub.max (ε)    in MeOH         240(36,900), 267(19,900), 286(sh),                    417(6,800)    in 0.1N HCl--MeOH                    240(32,200), 267(17,500), 286(sh),                    417(6,200)    in 0.1N NaOH--MeOH                    243(35,700), 282(sh), 324(8,000),                    518(5,500)    ______________________________________    Analysis        Found          Calcd for C.sub.22 H.sub.16 O.sub.8    ______________________________________                    C     64.86    C      64.71                    H     3.93     H      3.95    EI-MS (m/z)     408 (M.sup.+)    TLC, SiO.sub.2    CH.sub.2 Cl.sub.2 --MeOH (9:1)                    Rf    0.51    EtOAc--MeOH (4:1)     0.63    ______________________________________

                  TABLE 4    ______________________________________    .sup.1 H-NMR data of BU-3839T (400 MHz, in DMSO-d.sub.6)     ##STR7##    Chemical shift     Multiplicity    δ (ppm)               Proton  (J = Hz)      Assignments    ______________________________________    2.72       1H      dd (4.0 and 5.6)                                     H-17    2.86       1H      dd (2.8 and 5.6)                                     H-17    2.91       3H      s             H-13    3.64       1H      dd (2.8 and 4.0)                                     H-16    3.82       1H      dd (6.1 and 11.3)                                     H-15    4.21       1H      dd (6.1 and 11.3)                                     H-15    5.02       1H      t (6.1)       C-15 OH    5.72       1H      s             C-14 OH    6.52       1H      s             H-3    7.42       1H      d (8.1)       H-10    7.72       1H      d (7.3)       H-8    7.79       1H      dd (7.3 and 8.1)                                     H-9    7.97       1H      s             H-6    12.64      1H      s             C-11 OH    ______________________________________

Structure Determination

The UV absorptions and observed pH shifts of BU-3839T suggested a closeresemblance to those of the pluramycin group of antibiotics. The ¹ H-NMRspectrum revealed the presence of a total of 16 protons which wereanalyzed as 5 aromatic protons and 1 methyl, 2 methylene, 1 methine and3 hydroxy groups. The presence of these functionalities was ascertainedby the ¹³ C-NMR spectrum. The spectrum exhibited 3 carbonyl (178.0,181.2, 186.6 ppm), 14 sp² (with proton×5, 111.3, 118.6, 124.6×2, 136.5ppm and without proton×9, 116.7, 119.7, 125.7, 132.0, 135.6, 148.2,155.5, 161.3, 168.9 ppm) and 1 quaternary carbon (74.2 ppm) in additionto 1 methyl (23.1 ppm), 2 methylene (41.8, 65.1 ppm) and 1 methinecarbon (53.2 ppm) signals. Unlike those of the common pluramycin groupof antibiotics, the ¹ H- and ¹³ C-NMR spectra of BU-3839T did not showthe signals assignable to amino or neutral sugar. The molecular formulaassigned to the antibiotic (C₂₂ H₁₆ O₈) also showed the absence of anamino functionality in the molecule. The combined information, thus,suggested that BU-3839T had the chromophore structure of the pluramycinfamily of antibiotics. Three antibiotics with such type of structure,indomycinones, SF-2330 and SS-43405D and E have been reported. The ¹ H-and ¹³ C-NMR data described for SF-2330 were nearly identical with thoseof BU-3839T in the chromophore moiety and the differences appeared toreside only in the alkyl side chain of the 4-carbon unit (¹³ C-NMR:41.8t, 53.2 d, 65.1 t and 74.2 s). The behavior of UV maxima of BU-3839T andSF-2330 was consistent at different pHs. The ¹ H-NMR spectrum (inDMSO-d6) of the side chain moiety showed 7 protons, which were analyzedas two ABX's (2.72, 2.86 and 3.64 ppm and 3.82, 4.21 and 5.02 ppm), andan isolated hydroxyl proton (5.72 ppm). Upon D₂ O addition, the hydroxylproton and one of the lower-field ABX proton (5.02 ppm) disappeared withconcomitant collapsing of the ABX to a AB quartet. The spectroscopicresults taken together with the earlier physico-chemical data indicatedthe following structure for BU-3839T. ##STR8##

Biological Activity Antimicrobial Activity

Antimicrobial Activity of BU-3839T was tested against a variety ofbacteria and fungi by a two-fold serial dilution in agar media. As shownbelow in Table 5, BU-3839T exhibited remarkable inhibitory activityagainst gram-positive bacteria but relatively weak activity againstgram-negative and anaerobic bacteria. Yeast and fungi were notsusceptible to the antibiotic.

                  TABLE 5    ______________________________________    Antimicrobial activity                                    MIC    Test organism          Medium*  (mcg/ml)    ______________________________________    Staphylococcus aureus                     209P      NA       0.05    Staphylococcus aureus                     Smith     NA       0.05    Staphylococcus aureus                     A20234    NA       0.05    Staphylococcus epidermidis                     D153      NA       0.1    Streptococcus faecalis                     A9612     NA       0.4    Streptococcus pyogenes                     A20201    NA       0.1    Micrococcus luteus                     PCI 1001  NA       0.025    Bacillus subtilis                     PCI 219   NA       0.05    Escherichia coli NIHJ      NA       >50    Klebsiella pneumoniae                     D11       NA       6.3    Pseudomonas aeruginosa                     A9930     NA       >50    Proteus vulgalis A9436     NA       6.3    Proteus mirabilis                     A9554     NA       >50    Bacteroides fragilis                     A22035    GAM      0.1    Clostridium difficile                     A21675    GAM      0.4    Clostridium perfringens                     A9635     GAM      0.4    Preopionibacterium acnes                     A21933    GAM      0.8    Candida albicans IAM 4888  SDA      >50    Cryptococcus neoformans                     D49       SDA      >50    Aspergillus fumigatus                     IAM 2530  SDA      >50    Trichophyton mentagrophytes                     D155      SDA      >50    ______________________________________     *NA: Nutrient agar     GAM: Gifu anaerobic medium agar     SDA: Sabouraud dextrose agar

Antitumor activity

BU-3839T was tested for in vitro cytotoxicity against murine and humantumor cells and for in vivo antitumor activity in mice. Mitomycin C wasused as a reference compound in both in vitro and in vivo experiments.B16-F10 (murine melanoma) and Moser (human colorectal carcinoma) cellswere grown to the logarithmic phase in the enriched Eagle minimumessential medium (MEM) supplemented with fetal calf serum (FCS, 10%) andkanamycin (60 mcg/ml), and HCT-116 (human colon carcinoma) cells werealso grown to the logarithmic phase in Maccoy's 5A medium supplementedwith FCS (10%). penicillin (100 μ/ml) and streptomycin (100 mcg/ml). Thetumor cells were harvested and implanted into wells of the 96-wellmicrotiter plate with test materials at the inoculum sizes of 3.0×10⁴,6.0×10⁴ and 6.0×10⁴ cells/ml, respectively. They were incubated at 37°C. in humidified atmosphere of 5% CO₂ and 95% air for 72 hours. Thecytotoxic activities against the above tumor cell lines were determinedcolorimetrically at 540 nm after staining viable cells with neutral red.The results were summarized in Table 6. Cytotoxicities of BU-3839T werequite potent against these tumor cells and were approximately 300-600times superior to those of mitomycin C in terms of IC₅₀ value.Inhibitory effects of BU-3839T on macromolecule (DNA, RNA and protein)synthesis were determined in vitro. Cultured L1210 murine leukemia cells(5×10⁵ cells/ml) were incubated with test materials at 37° C. for 15min. Labelled precursor, ³ H-thymidine, ¹⁴ C-uridine or ³ H-leucine wasadded into the cultured mixtures and further incubated for 60 min. Afterwashing with chilled 5% trichloroacetic acid solution, the radioactivityincorporated into the acid-insoluble fraction of the tumor cells wasdetermined in a liquid scintillation counter. As shown in Table 7, incontrast to mitomycin C which showed specific inhibition of DNAsynthesis, BU-3839T inhibited DNA, RNA and protein synthesisnon-specifically. In vivo antitumor activity of BU-3839T was tested inthe experimental mouse tumor systems. Female CDF₁ and male BDF₁ micewere intraperitoneally inoculated with 0.4 ml of diluted ascitic fluidcontaining 10⁶ lymphocytic leukemia P388 cells and 0.5 ml of 10%melanotic melanoma B16 brei, respectively. Test compounds wereadministered intraperitoneally by the following dosing schedules; once aday on days 1, 2 and 3 (QD×3) or on days 1, 5 and 9 (Q4D×3). As shown inTables 8 and 9, BU-3839T demonstrated relatively broad and moderatechemotherapeutic activity against P388 leukemia with maximum T/C valueof 145%, whereas it showed minimum antitumor activity against ip-B16melanoma at the doses tested.

                  TABLE 6    ______________________________________    In vitro cytotoxicities against murine and human    tumor cells             IC.sub.50 (mcg/ml)    Compound   B16-F10      Moser   HCT-116    ______________________________________    BU-3839T   0.0009       0.0046  0.0026    Mitomycin C               0.50         1.2     0.80    ______________________________________

                  TABLE 7    ______________________________________    Inhibition of macromolecule synthesis in L1210    leukemia cells              IC.sub.50 (mcg/ml)    Compound    DNA         RNA     Protein    ______________________________________    BU-3839T    0.013       0.029   0.039    Mitomycin C 1.7         >100    >100    ______________________________________

                  TABLE 8    ______________________________________    Antitumor activity against P388 leukemia (ip)                                        Body weight              Dose*.sup.1                         MST*.sup.2                                   T/C  change on    Compound  (mg/kg/day)                         (day)     (%)  day 4 (g)    ______________________________________    BU-3839 T 1.0         7.0      70.sup.                                        -2.5              0.5        14.5      145*.sup.3                                        -2.3              0.25       12.5      125*.sup.3                                        -1.8              0.13       14.0      140*.sup.3                                        -0.5              0.063      13.0      130*.sup.3                                        +0.8              0.031      12.5      125*.sup.3                                        +1.3              0.016      11.5      115  +1.0              0.008      11.0      110  +1.0    Mitomycin C              2          20.0      200*.sup.3                                        -1.8              1          14.5      145*.sup.3                                        -0.3              0.5        15.0      150*.sup.3                                        -0.5              0.25       13.0      130*.sup.3                                        +1.5    Vehicle   --         10.0      --   +1.4    ______________________________________     *.sup.1 QD × 3, ip     *.sup.2 Median survival time     *.sup.3 Significant antitumor effect (T/C ≧ 125%)

                  TABLE 9    ______________________________________    Antitumor activity against B16 melanoma (ip)                                        Body weight              Dose*.sup.1                         MST*.sup.2                                   T/C  change on    Compound  (mg/kg/day)                         (day)     (%)  day 5 (g)    ______________________________________    BU-3839 T 2.0        Tox       Tox  --              1.0        10.0      74.sup.                                        -3.3              0.5        11.0      81.sup.                                        -3.8               0.25      17.0      126*.sup.3                                        -0.5               0.13      15.5      115  +0.5               0.063     16.5      122  +0.8    Mitomycin C              2.0        30.0      222*.sup.3                                        +0.5              1.0        20.5      152*.sup.3                                        +1.0              0.5        18.0      133*.sup.3                                         0.0               0.25      15.0      111  +0.8    Vehicle   --         13.5      --   +1.0    ______________________________________     *.sup.1 Q4D × 3, ip     *.sup.2 Median survival time     *.sup.3 Significant antitumor effect (T/C ≧ 125%)

As shown above BU-3839T possesses potent antibacterial activity and isthus useful in the therapeutic treatment of mammals and other animalsfor diseases caused by such organisms. Additionally, the compound may beutilized for other conventional in vitro applications of antibacterialagents such as disinfecting medical and dental equipment.

The in vitro and in vivo antitumor data presented above indicate thatBU-3839T is also therapeutically useful in inhibiting the growth ofmalignant tumors in mammalian hosts.

The present invention, therefore, provides a method for therapeuticallytreating an animal host affected by a bacterial infection, whichcomprises administering to said host an effective antibacterial dose ofBU-3839T, or a pharmaceutical composition thereof.

Also provided is a method for inhibiting the growth of malignant tumorsin mammals which comprises administering to said mammalian host aneffective tumor-inhibiting dose of BU-3839T, or a pharmaceuticalcomposition thereof.

In another aspect the present invention provides a pharmaceuticalcomposition which comprises an effective antibacterial amount ofBU-3839T in combination with an inert pharmaceutically acceptablecarrier or diluent.

Additionally, the invention provides a pharmaceutical composition whichcomprises an effective tumor-inhibiting amount of BU-3839T incombination with an inert pharmaceutically acceptable carrier ordiluent.

The pharmaceutical compositions may contain other active antibacterialor antitumor agents and may be made up in any pharmaceutical formappropriate for the desired route of administration. Examples of suchcompositions include solid compositions for oral administration such astablets, capsules, pills, powders and granules, liquid compositions fororal administration such as solutions, suspensions, syrups or elixersand preparation for parenteral administration such as sterile solutions,suspensions or emulsions. They may also be manufactured in the form ofsterile solid compositions which can be dissolved in sterile water,physiological saline or some other suitable sterile injectable mediumimmediately before use.

For use as an antimicrobial agent, the BU-3839T or pharmaceuticalcomposition thereof, is administered so that the concentration of activeingredient is greater than the minimum inhibitory concentration for theparticular organism being treated. For use as an antitumor agent,optimal dosages and regiments of BU-3839T for a given mammalian host canbe readily ascertained by those skilled in the art. It will, of course,be appreciated that the actual dose of compound used will vary accordingto the particular composition formulated, the mode of application andthe particular situs, host and disease being treated. Many factors thatmodify the action of the drug will be taken into account including age,weight, sex, diet, time of administration, route of administration, rateof excretion, condition of the patient, drug combinations, reactionsensitivities and severity of the disease.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention. Unless otherwiseindicated all volume ratios indicated below are volume/volume.

EXAMPLE 1 Fermentation of BU-3839T

A loopful of the slant culture of Steptomyces violaceus strain No.P950-4 was inoculated into a 500-ml Erlenmeyer flask containing 100 mlof the seed medium consisting of 2% glucose, 1% Pharmamedia (Traders)and 0.5% CaCO₃, the pH being adjusted to 7.0 before autoclaving. Thefermentation was carried out at 28° C. for 4 days on a rotary shaker(200 rpm) and 5 ml of broth was transferred into a 500-ml Erlenmeyerflask containing 100 ml of production medium having the same compositionas the seed medium. The fermentation was carried out for 3 days underthe same conditions as the seed culture. The antitumor activity infermentation broths was determined by the paper disc agar diffusionassay using Bacillus subtilis M45 (rec⁻) as the test organism and invitro cytotoxic activity against B16 melanoma cells. The antibioticpotency of 7 μg/ml was obtained after three days cultivation.

The fermentation was also carried out in a jar fermentor. A 500 mlportion of the seed culture obtained by the flask fermentation wastransferred into a 20 liter jar fermentor (Marubishi MSJ-20) containing12 liters of production medium having the same composition as the flaskfermentation. The fermentation was run at 28° C. with agitation at 250rpm and aeration rate of 12 liters per minute. The antibiotic productionreached a maximum of 12 μg/ml after 40-50 hrs fermentation.

EXAMPLE 2 Isolation and Purification of BU-3839T

The whole harvested broth (50 L, pH 8.4) as produced in Example 1 wasextracted with n-butanol (25 L) under vigorous stirring. The separatedn-butanol phase was evaporated under reduced pressure to an aqueoussolution (1 L) which was extracted three times with 1 L each of ethylacetate. The organic extracts were combined and concentrated in vacuo to200 ml of the solution. The solution was added dropwise to 2.3 L ofn-hexane under stirring. The precipitates deposited were collected byfiltration and dried in vacuo to yield the crude solid of BU-3839T (6.9g).

The crude solid was dissolved in methylene chloride (50 ml) and appliedon a column of silica gel (φ5.0×50 cm) which had been pre-washed withmethylene chloride. The elution was carried out with methylene chlorideand an increasing amount of methanol (99.5:0.5-98:2). The eluate waschecked by the bioassay using Bacillus subtilis M45 rec⁻ mutant) and theactive fractions eluted with methylene chloride-methanol (99:1-98:2)were pooled and concentrated in vacuo. Upon standing in a cold room, theconcentrate deposited yellowish-orange needles of pure BU-3839T (109mg). Rechromatography of the mother liquor and the subsequent activeeluate afforded a further amount of pure BU-3839T (120 mg).

We claim:
 1. A process for the preparation of BU-3839T having theformula ##STR9## which comprises cultivating a BU-3839T-producing strainof Streptomyces violaceus in an aqueous nutrient medium containingassimilable sources of carbon and nitrogen under submerged aerobicconditions until a substantial amount of BU-3839T is produced by saidorganism in said culture medium and then recovering said BU-3839T fromthe culture medium.
 2. The process according to claim 1 wherein theBU-3839T-producing strain is Streptomyces violaceus ATCC 53807 or avariant or mutant thereof.